Mycobacterium paratuberculosis causes Johne's disease (paratuberculosis) in dairy cattle. The disease is characterized by chronic diarrhea, weight loss, and malnutrition, resulting in estimated losses of $220 million per year in the USA alone. World-wide, the prevalence of the disease can range from as low as 3-4% of the examined herds in regions with low incidence (such as England), to high levels of 50% of the herds in some areas within the USA (Wisconsin and Alabama). Cows infected with Johne's disease are known to secrete Mycobacterium paratuberculosis in their milk. In humans, M. paratuberculosis bacilli have been found in tissues examined from Crohn's disease patients indicating possible zoonotic transmission from infected dairy products to humans.
Unfortunately, the virulence mechanisms controlling M. paratuberculosis persistence inside the host are poorly understood, and the key steps for establishing the presence of paratuberculosis are elusive. Mechanisms responsible for invasion and persistence of M. paratuberculosis inside the intestine remain undefined on a molecular level (Valentin-Weigand and Goethe, 1999, Microbes & Infection 1: 1121-1127). Both live and dead bacilli are observed in sub-epithelial macrophages after uptake. Once inside the macrophages, M. paratuberculosis survive and proliferate inside the phagosomes using unknown mechanisms.
M. paratuberculosis is closely related to Mycobacterium avium subspecies avium (hereinafter referred to as Mycobacterium avium or M. avium), which is a persistent health problem for immunocompromised humans, particularly HIV-positive individuals. Limited tools are available to researchers to definitively identify M. paratuberculosis and to distinguish it from M. avium. Existing methods are subject to high cross-reactivity, poor sensitivity, specificity, and predictive value. This dearth of knowledge translates into a lack of suitable vaccines for prevention and treatment of Johne's disease in animals, and of Crohn's disease in humans.
The current challenge in screening M. paratuberculosis is to identify those targets that are essential for survival of the bacilli during infection. Recently, random transposon mutagenesis-based protocols were employed for functional analysis of a large number of genes in M. paratuberculosis (Harris et al., 1999, FEMS Microbiology Letters 175: 21-26; Cavaignac et al., 2000, Archives of Microbiology 173: 229-231). When M. paratuberculosis was used as a target for mutagenesis, the libraries were screened to identify auxotrophs or genes responsible for survival under in vitro conditions. In these reports, six auxotrophs and two genes responsible for cell wall biosynthesis were identified (Harris et al., 1999; Cavaignac et al., 2000). So far, none of these libraries have been screened for virulence determinants.
Many clinical methods for detecting and identifying Mycobacterium species in samples require analysis of the bacterium's physical characteristics (e.g., acid-fast staining and microscopic detection of bacilli), physiological characteristics (e.g., growth on defined media) or biochemical characteristics (e.g., membrane lipid composition). These methods require relatively high concentrations of bacteria in the sample to be detected, may be subjective depending on the clinical technician's experience and expertise, and are time-consuming. Because Mycobacterium species are often difficult to grow in vitro and may take weeks to reach a useful density in culture, these methods can also result in delayed patient treatment and costs associated with isolating an infected individual until the diagnosis is completed.
More recently, assays that detect the presence of nucleic acid derived from bacteria in the sample have been preferred because of the sensitivity and relative speed of the assays. In particular, assays that use in vitro nucleic acid amplification of nucleic acids present in a clinical sample can provide increased sensitivity and specificity of detection. Such assays, however, can be limited to detecting one or a few Mycobacterium species depending on the sequences amplified and/or detected.
The genome sequences of both M. avium (Institute for Genomic Research) and of M. paratuberculosis (Li et al., 2005, Proc. Natl. Acad. Sci. USA 102: 12344-12349; GenBank accession No. AE016958) are currently available. It would be useful to analyze these genomes to provide a higher resolution analysis of M. avium subspecies genomes. A better understanding of the virulence mechanisms and pathogenesis of M. paratuberculosis is required to develop more effective vaccine and chemotherapies directed against M. paratuberculosis. 
In view of the problems with bacterial specificity, the present inventors have focused their attention on identification of putative virulence factors that may contribute to the pathogenicity of M. paratuberculosis. This information could be used to design vaccines against pathogenic subspecies of M. avium. Such vaccines can be used for prevention and treatment of Johne's disease in animals or Crohn's disease in humans.